Anti phospho histone h3

Embryos were collected at stage 16 and fixed with paraformaldehyde. The frozen samples were sectioned in 10 μm and the sections were stained with 1:200 Anti-phospho-histone H3 (Millipore 16–657) to detect cell proliferation and 300 nM DAPI (Life Technologies D1306) for nuclear conterstain. Whole embryos at stage 16 were lysed with 1%triton-X100 in PBS containing a protease inhibitor cocktail (Roche) and the samples were subject to Western blot analysis with activated caspase3 antibody(Abcam 13847) to check cell apoptosis.

Immunostaining was done as previously described. Briefly, appropriately aged guts were dissected in PBS. The dissected guts were immediately fixed in glutamate buffer containing 4% Formaldehyde for 20 minutes followed by a series of methanol washes as previously described [37 (link)]. The fixed guts were washed in PBS containing 0.1% Triton X-100 and 0.5% BSA (Gut Buffer). It was then blocked-in gut buffer for one hour followed by overnight incubation at 4°C in appropriate antibody. Secondary antibodies were used at 1:500. After the secondary antibody treatment, the guts were mounted on slides with Mowiol/Dabco solution. For pH3 staining the guts were fixed for 45 minutes in PBS with 4% formaldehyde.
The following primary antibodies were used: Sox21a antibody was previously generated in the lab. The anti-Delta (C594.9B), anti-Prospero (MR1A), anti-β-galactosidase (40-1a) were obtained from the Developmental Studies Hybridoma Bank (DSHB) and the Anti-phospho-Histone H3 (06–570) from Millipore. Anti-GFP was obtained from Thermofisher Scientific and anti-mCherry was obtained from Biovision. Anti-Imp was a gift from P. Macdonald. Anti-Lin28 was a gift from N. Sokol. Fluorescent secondary antibodies were obtained from Jackson Immunoresearch. Hoechst 33258 (Sigma Aldrich) was used to stain DNA.

Female fly intestines were dissected in PBS 1X solution and then fixed in Fixation Buffer containing 100 mM glutamic acid, 25 mM KCl, 20 mM MgSO4, 4 mM sodium phosphate, 1 mM MgCl2, and 4% formaldehyde for 45 minutes at room temperature. Samples were blocked using Blocking buffer containing PBS 1X, 0.5% BSA, and 0.1% Triton X-100. Samples were then incubated in the same buffer containing primary antibodies over-night at 4°C [anti-phospho-Histone H3 from Millipore (1:2000), anti-Beta-Galactosidase from DHSB (1:500), anti-Prospero from DHSB (1:200)]. Fluorescent secondary antibodies were obtained from Jackson Immunoresearch. DNA was stained using Hoechst and Visceral muscle was stained using Alexa Fluor 647 Phalloidin (from Invitrogen, 1:400). For Delta (from DHSB, 1:500), Sox21a (generated in the lab [43 (link)], 1:5000), p4EBP (from Cell signaling technologies, 1:200), zfh2 (kindly provided by Chris Doe, 1:200) and PDM1 (kindly provided by Xiaohang Yang and Cai Yu, 1:1000) staining samples were fixed in Fixation Buffer and Heptane, dehydrated with 100% Methanol and progressively re-hydrated in blocking Buffer. Confocal imaging was done using the Leica SP5 system and processed with Adobe Photoshop CS6.